This invention is directed to test strips for determination of characteristics of samples, unitized housings, and kits incorporating the test strips and housings, and methods of determining the characteristics of samples using the test strips and housings.
Among the many analytical systems used for detection and/or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems. Among the analytes frequently assayed with such systems are:
(1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed as a marker of human pregnancy; PA1 (2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus, hepatitis virus, and Giardia; PA1 (3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibody to the bacterium Helicobacter pylori and to human immunodeficiency virus (HIV); PA1 (4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; PA1 (5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; PA1 (6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, and marijuana; PA1 (7) environmental pollutants such as pesticides and aromatic hydrocarbons and PA1 (8) vitamins. PA1 (1) at least two substantially planar opposable components, wherein one of the substantially planar components has on its surface a chromatographic medium; and PA1 (2) means for opposing the opposable components and applying pressure thereto, the pressure being sufficient to transfer fluid from one opposable component to another opposable component in a direction substantially normal to the opposable component so that the sample is applied to the chromatographic medium for detection and/or determination of the analyte thereon. PA1 (1) at least three substantially planar opposable components, wherein one of the substantially planar components has on its surface a chromatographic medium, the chromatographic medium having first and second ends; PA1 (2) means for opposing the opposable components pairwise in at least two different combinations and applying pressure thereto, the pressure being sufficient to transfer fluid from one opposable component to another in a direction substantially normal to the opposable components so that the sample is applied to the chromatographic medium and flows through the chromatographic medium from the first end to the second end for detection and/or determination of the analyte on the chromatographic medium; and PA1 (3) at least one applicator and one absorber located on one of the opposable components and positioned in such manner that, when the opposable component on which the applicator and the absorber is located is brought into opposition to the opposable component on which the chromatographic medium is located, a second liquid is applied to the chromatographic medium and flows through the chromatographic medium from the second end to the first end, thereby reversing the flow through the chromatographic medium, with the detection and/or determination of the analyte being made subsequent to reversal of the flow through the chromatographic medium. PA1 (1) a first opposable component including a sample preparation zone adapted to receive a sample to be assayed; and PA1 (2) a second opposable component including a chromatographic medium. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a sample preparation zone for receiving a sample to be tested. PA1 (1) applying the aqueous sample to the sample application pad of the chromatographic assay device containing a resolubilizable labeled first specific binding partner on the detector application pad; PA1 (2) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample comprises an aqueous liquid resolubilizing the labeled specific binding partner in the detector application pad, and such that the sample and the resolubilized labeled specific binding partner are applied to the conductor; PA1 (3) allowing the sample and labeled specific binding partner to move through the conductor and then through at least a portion of the chromatographic medium so that the labeled specific binding partner gives a detectable indication of the presence and/or quantity of the analyte in the test sample; and PA1 (4) observing and/or measuring the labeled specific binding partner in at least a portion of the chromatographic medium in order to detect and/or determine the analyte. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (1) the sample application pad applying the sample to the detector application pad and thus to the first end of the chromatographic medium through the conductor; and PA1 (2) the absorber being in operable contact with the second end of the chromatographic medium. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (1) the sample to be tested to be applied to the detector application pad and then to the first end of the chromatographic medium; and PA1 (2) the absorber to be brought into operable contact with the second end of the chromatographic medium. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (1) results in the conductor coming in operable contact with the first applicator, results in the conductor coming in operable contact with the second applicator, and results in the second applicator coming in operable contact with the first end of the chromatographic medium, thereby placing the first and second applicator in operable contact with each other to apply the contents of the first and second applicator of the chromatographic medium; and PA1 (2) results in the absorber coming in operable contact with the second end of the chromatographic medium. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including an applicator divided into two sectors: PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a first applicator containing a first specific binding partner to the analyte in a form that can be resolubilized by addition of a first aqueous liquid to the first applicator; and PA1 (3) a third opposable component including: PA1 (1) applying the sample to the first applicator of the chromatographic assay device, the sample comprising the first aqueous liquid; PA1 (2) applying a reconstitution fluid to the second applicator, the reconstitution fluid comprising the second aqueous liquid; PA1 (3) bringing the first and second opposable components of the chromatographic assay device into opposition, such that the sample and the resolubilized first specific binding partner of the analyte are applied to the first conductor and then to the first end of the chromatographic medium; PA1 (4) allowing a sample and a resolubilized first specific binding partner to move through at least a portion of the chromatographic medium blocking binding sites on the immunological analogue immobilized in the discrete area; PA1 (5) separating the first and second opposable components so that they are no longer in opposition; PA1 (6) bringing the first and third opposable components into opposition such that the resolubilized labeled second specific binding partner is applied to the second conductor and then to the second end of the chromatographic medium; PA1 (7) allowing the resolubilized labeled second specific binding partner to move through at least a portion of chromatographic medium overlapping the entire chromatographic medium through which the sample and the resolubilized first specific binding partner is drawn so that, in the presence of analyte in the test sample, the labeled second specific binding partner binds to the analyte or immunological analogue thereof immobilized in the discrete area due to the binding of sample and analyte with the first specific binding partner; and PA1 (8) observing and/or measuring the second specific binding partner in the discrete area to detect and/or determine the analyte. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (1) a first opposable component comprising a chromatographic medium having a first end and a second end and having immobilized thereon, in separate discrete non-overlapping areas, each area being substantially smaller than the area of the chromatographic medium; PA1 (2) a second opposable component including a first applicator containing a first specific binding partner to the analyte in a form that can be resolubilized by the addition of an aqueous sample to the first applicator, the first specific binding partner being covalently linked to biotin, the first specific binding partner not capable of being bound by the secondary specific binding partner; PA1 (3) a third opposable component including: PA1 (1) a first opposable component including: PA1 (2) a second opposable component including an applicator containing a specific binding partner to the analyte in a form that can be resolubilized by addition of an aqueous liquid to the applicator. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including: PA1 (3) a third opposable component including a second absorber.
Such chromatographic systems are frequently used by physicians and medical technicians for rapid in-office diagnosis and therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home monitoring of such conditions and disorders.
Among the most important of such systems are the "thin layer" systems in which a solvent moves across a thin, flat absorbent medium.
Among the most important of tests that can be performed with such thin layer systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody. The use of immunoassays as a means of testing for the presence and/or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (Singer et al., Am. J. Med. 22:888-892 (1956)).
Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immunochromatography. In general, this technique uses a disclosing reagent or particle that has been linked to an antibody to the molecule to be assayed, forming a conjugate. This conjugate is then mixed with a specimen and, if the molecule to be assayed is present in the specimen, the disclosing reagent-linked antibodies bind to the molecule to be assayed, thereby giving an indication that the molecule to be assayed is present. The disclosing reagent or particle can be identifiable by color, magnetic properties, radioactivity, specific reactivity with another molecule, or another physical or chemical property. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested.
Immunochromatographic assays fall into two principal categories: "sandwich" and "competitive," according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. The antigen to be detected can itself be an antibody, such as in serological assays for H. pylori-specific antibody. In such cases, the antibody to be detected can be bound to a specific antigen. Alternatively, the antigen to be detected can be detected indirectly by using a labeled second antibody that binds to the first antibody to the analyte to be detected.
In general, the sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a label or a disclosing reagent, such as dyed latex, a colloidal metal sol, or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or zone of immobilized antibodies to the analyte of interest. The chromatographic medium often is in the form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs and the bound labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results.
Examples of sandwich immunoassays performed on test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al. both of which are incorporated herein by this reference.
In competitive immunoassays, the label is typically a labeled analyte or analyte analogue which competes for binding of an antibody with any unlabeled analyte present in the sample. Competitive immunoassays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which are incorporated herein by this reference.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal samples, contain particulate matter that can clog the pores of the chromatographic medium, greatly hindering the immunochromatographic process. Other samples, such as blood, contain cells and colored components that make it difficult to read the test. Even if the sample does not create interference, it is frequently difficult with existing chromatographic test devices to apply the sample to the chromatographic medium so that the sample front moves uniformly through the chromatographic medium to insure that the sample reaches the area where binding is to occur in a uniform, straight-line manner.
Other problems exist with currently-available test strips because of the nature of the sample to be assayed or the assay to be carried out. With such devices, it is impractical to perform washing steps which are frequently desirable to improve sensitivity and to reduce background. Also, it is difficult, and in many cases impossible, to carry out preincubation steps within the device.
Additionally, there is a need for an immunochromatographic assay device that can carry out a broad range of separations, such as the separation of fat from milk or the separation of organic chemicals such as the separation of benzene from toluene.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, such as AIDS and hepatitis, has exacerbated these problems. It is rarely possible to apply a sample (such as feces) or a sampling device (such as a throat swab) directly to the chromatographic medium. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. These reactions are typically carried out by the physician or technician performing the test in several small vessels, such as test tubes, or microfuge tubes, requiring the use of transfer devices, such as pipettes. Each of these devices is then contaminated and must be disposed of using special precautions so that workers or people who may inadvertently come into contact with the waste do not become contaminated.
Still another limitation on chromatographic devices currently available for use by the clinician or technician is their inability to perform two-directional or two-dimensional chromatography. These techniques have long been known to be powerful analytical tools, but their complexity relative to simple unidirectional chromatography has made it difficult to apply them to test strip devices in the physician's office or a clinical laboratory.
Accordingly, there is a need for an improved assay device capable of handling a broad range of chromatographic assays. Such a device should be able to handle all types of immunoassays, including both sandwich and competitive immunoassays as well as other types of assays using chromatography. Such a device should be capable of receiving a possibly contaminated sample or a sample preparation device directly so as to eliminate the need for extraction vessels and transfer devices. Such a device, preferably in the form of a test strip, should also be capable of performing immunochromatographic assays on colored samples or samples containing particulates without interference and should be able to deliver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the tests. Additionally, such an improved test strip should be capable of performing two-directional or two-dimensional chromatography when used in clinical laboratories or physicians' offices.